Sublancin lantibiotic produced by Bacillus subtilis 168

ABSTRACT

An antimicrobial peptide produced by  Bacillus subtilis  168 was isolated and characterized and named sublancin 168. The invention includes DNA encoding for the sublancin 168 peptides and peptides which are at least 80% identical to the sublancin 168 peptide. The peptides may be administered as anti-bacterials, or may be used in food preservation. The peptides may also be co-administered with other lantibiotics (such as nisin and subtilin), or with known antibiotics.

FIELD OF THE INVENTION

[0001] The invention relates to novel bacterially-produced antimicrobialpeptides; more particularly the invention relates to adehydroalanine-containing lantibiotic.

BACKGROUND OF THE INVENTION

[0002] Lantibiotics are bacterially-produced antimicrobial peptides thatpossess unique chemical and biological properties owing to theircontaining a variety of unusual amino acid residues. Lantibiotics aredefined as such by the presence of lanthionine or β-methyllanthionine,which are introduced by a posttranslational process in which serine orthreonine is dehydrated to the corresponding dehydro residue, which thenreacts in a Michael-type addition of a cysteine sulfhydryl group to thedouble bond of the dehydro residue to form a thioether link [reviewed in(1-6)]. Mature lantibiotics typically contain one or more dehydroresidues that do not participate in lanthionine bridges. The uniqueproperties that are conferred by these unusual residues results in theirbeing useful components in the design of novel biomolecules (1,2,7,8).

[0003] One of the attractive features of lantibiotics is that they arecomprised of gene-encoded polypeptide sequences, so their structures canbe manipulated by protein engineering. Whereas this is simple inconcept, putting it into practice requires the utilization of manydifferent genetic and recombinant DNA techniques, including the removaland replacement of chromosomal segments with theirgenetically-engineered counterparts. Ideally, these manipulations needto be done in such a way that the engineered lantibiotic analog beefficiently produced so that useful amounts of the analog are availablefor experimentation, which implies a need to engineer regulatoryelements. Only a few bacterial strains have been sufficientlycharacterized to permit these manipulations to be performed in aconvenient and facile manner. One such well-characterized bacterialstrain is Bacillus subtilis 168, which is second only to E. coli in theextent to which tools of genetic and protein engineering have beendeveloped, which has contributed to the extensive use of B. subtilis 168for the industrial production of bio-engineered materials. The advantageof B. subtilis 168 over other bacterial strains has recently beenincreased even more by the availability of the complete sequence of theB. subtilis 168 genome (9).

SUMMARY OF THE INVENTION

[0004] The present inventor has discovered a new lantibiotic, namedsublancin 168, that is a natural product produced by B. subtilis 168.Although approximately twenty lantibiotics are already known, the factthat this new lantibiotic is endogenous to B. subtilis 168, and thus canbe studied and manipulated using the powerful methods that are availablein this strain, suggests that progress in our understanding oflantibiotics will be accelerated by our ability to study and manipulatesublancin and the genes associated with its production in its natural B.subtilis 168 host. In addition to this practical aspect of thediscovery, sublancin 168 has structural features and physicalproperties, such as the presence of disulfide bridges and extraordinarystability, that are unprecedented among the known lantibiotics.

[0005] Therefore, the present invention is directed to a peptide havingan amino acid sequence which is at least 80% identical with SEQ ID NO: 7prior to dehydration of serines and threonines and formation ofthioether cross-linkages.

[0006] The invention also is directed to a peptide having an amino acidsequence which is at least 80% identical with SEQ ID NO: 5 prior todehydration of serines and threonines and formation of thioethercross-linkages.

[0007] The invention is further directed to a peptide having an aminoacid sequence which is at least 80% identical with SEQ ID NO: 18.

[0008] The peptides of the invention may be incorporated into apharmaceutical preparation suitable for treating a bacterial infection.In addition, a bacterial-growth-inhibiting effective amount of one ormore of the peptides of the invention may be added to a food forpreservation against bacteria-mediated spoilage of the food.

[0009] The invention also includes a DNA which has a nucleic acidsequence which is at least 80% identical with SEQ ID NO: 4, preferablyat least 80% identical with nucleotides 219-389 or nucleotides 447-782of SEQ ID NO: 4, which includes the genetic sequence encoding thesublancin 168 peptide of the invention. More preferably, the DNA has anucleic acid sequence which is at least 80% identical with nucleotides219-389 of SEQ ID NO: 4.

[0010] The invention further includes a composition suitable for killingor inhibiting growth of bacteria, comprising the peptides of theinvention and a carrier. Another composition encompassed by theinvention comprises the peptides of the invention and a second activeagent selected from the group consisting of a lantibiotic (such as nisinor subtilin) and an antibiotic.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the active peak of sublancin recovered from thesupernatant using a hydrophobic interaction column and purified tonear-homogeneity on a reversed-phase HPLC column. The active peak showedabsorbances at 214, 254, and 280 nm.

[0012]FIG. 2 shows the sequence of presublancin 168. The P indicates thelocation of a consensus prokaryotic promoter site as described in thelegend of FIG. 3.

[0013]FIG. 3 shows the nucleotide sequence of the sublancin gene. Thecomplete coding sequences of the sunA and sunT genes and theirconceptual translations are available as Accession Number AF069294 inGenBank.

[0014]FIG. 4 shovers the alignment of pre-sublancin with Type AI andType All pre-lantibiotics. Conserved leader segments of AI and AII asidentified by Nes and Tagg (3). Sublancin shows homologies that arecharacteristic of Type AII lantibiotics, including the “diglycine motif”found in leaders that are normally cleaved by dual-function transportersthat contain a leader peptidase function (20), as described in the text.

[0015]FIG. 5 shows homologies of the N-terminal end of SunT to PepT andLcnDR3, which are lantibiotic and dual-function transportersrespectively.

[0016]FIG. 6 shows the proton NMR spectrum of sublancin 168.

[0017]FIG. 7 shows the locations of thioether and disulfide bridges insublancin 168.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention is directed to a peptide having an aminoacid sequence which is at least 80% identical with SEQ ID NO: 7 prior todehydration of serines and threonines and formation of thioethercross-linkages. Preferably, the peptide contains the amino acids ofpositions 7, 14, 16, 19, 22, 29 and 36 of SEQ ID NO: 7. It is morepreferred that a thioether cross-linkage is formed between the aminoacids of positions 19 and 22 and disulfide cross-linkages are formedbetween the amino acids of positions 7 and 36 and positions 14 and 29.The peptide may preferably have an amino acid sequence which is at least90% identical with SEQ ID NO: 7, most preferably, the amino acidsequence is 100% identical with SEQ ID NO: 7.

[0019] The invention is also directed to a peptide having an amino acidsequence which is at least 80% identical with SEQ ID NO: 5 prior todehydration of serines and threonines and formation of thioethercross-linkages. Preferably, the amino acid sequence is at least 90%identical with SEQ ID NO: 5, most preferably, the amino acid sequence is100% identical with SEQ ID NO: 5.

[0020] The invention is further directed to a peptide having an aminoacid sequence which is at least 80% identical with SEQ ID NO: 18.Preferably, the peptide contains the amino acids of positions 7, 14, 16,19, 22, 29 and 36 of SEQ ID NO: 18. It is further preferred that athioether cross-linkage is formed between the amino acids of positions19 and 22 and disulfide cross-linkages are formed between the aminoacids of positions 7 and 36 and positions 14 and 29. The amino acidsequence is preferably at least 90% identical with SEQ ID NO: 18, mostpreferably 100% identical with SEQ ID NO: 18.

[0021] The invention also includes a DNA which has a nucleic acidsequence which is at least 80% identical with SEQ ID NO: 4, preferablyat least 80% identical with nucleotides 219-389 or nucleotides 447-782of SEQ ID NO: 4, which includes the genetic sequence encoding thesublancin 168 peptide of the invention. More preferably, the DNA has anucleic acid sequence which is at least 80% identical with nucleotides219-389 of SEQ ID NO: 4. The DNA sequence is preferably at least 90%identical with SEQ ID NO: 4 or the portions thereof disclosed above(i.e., nucleotides 219-389 and/or nucleotides 447-782), most preferably100% identical.

[0022] All percentage identities for the amino acid and DNA sequencesnoted above can be determined using a variety of algorithms known in theart. An example of a useful algorithm in this regard is the algorithm ofNeedleman and Wunsch, which is used in the “Gap” program by the GeneticsComputer Group. This program finds the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps. Another useful algorithm is the algorithm of Smith andWaterman, which is used in the “BestFit” program by the GeneticsComputer Group. This program creates an optimal alignment of the bestsegment of similarity between two sequences. Optimal alignments arefound by inserting gaps to maximize the number of matches using thelocal homology algorithm of Smith and Waterman. It is preferred to usethe algorithm of Needleman and Wunsch to compare amino acid and DNApercentage identity in the present case to another amino acid or DNAsequence.

[0023]FIG. 4 compares presublancin with the Type A lantibiotics, whichare divided into two sub-types, AI and AII. The Type A lantibioticsinclude those that are the most thoroughly studied, such as nisin A,subtilin, epidermin, and Pep5. Type A lantibiotics are characterized bybeing elongated and cationic with molecular masses ranging from 2,151 to4,635 Da (1). The mature region of the sublancin peptide is cationic,and its predicted molecular mass is approximately 3,900 Da (depending onwhat posttranslational modifications have occurred), and thus possessescharacteristics of a Type A lantibiotic. Type AI and Type AIIlantibiotics differ in their leader segments, with the AII leaderscontaining a GA/GS/GG (“double-glycine”) sequence motif immediatelypreceding the cleavage site, and conserved EL/EV and EL/EM sequencesupstream from the cleavage site. Double-glycine-type leader peptides areunrelated to the N-terminal sequences utilized by the sec pathway, andthe corresponding ABC transporters typically possess a dual functionthat both removes the leader peptide and translocates it across thecytoplasmic membrane (20). These features are shared by severalnon-lantibiotic antimicrobial peptides, including pediocin andlactococcin A, which are produced by gram-positive bacteria; and bycolicin V which is produced by gram-negative E. coli (20), suggestingthat the double-glycine leader peptide may represent an evolutionarybranch-point between the lantibiotic and non-lantibiotic peptides.

[0024] If the double-glycine leader peptide of sublancin is cleaved by aprotease that is a component of a dual-function transporter, then thetransporter should contain an identifiable protease domain. Examinationof the ORF that is immediately downstream from the sunA gene shows thatthe putative SunT protein shows such a protease domain. FIG. 5 comparesSunT with two other ABC-transporter proteins; PepT, which is thetransporter for the Type AI lantibiotic Pep5 (which does not have adiglycine-type leader peptide), and with LcnDR3, which is thetransporter for the non-lantibiotic lactococcin DR (which does have adiglycine-type leader peptide). The LcnDR3 protein contains anN-terminal protease domain that consistently appears in thedual-function transporters that cleave the leaders that-contain thediglycine motif (20), and this protease domain also appears in the SunTprotein, and contains-the conserved cysteine and histidine residues thatare part of the active site of the proteolytic domain.

[0025] The fact that the leader segment of sublancin contains thatconserved features that are typical of Type AII lantibiotics constitutesevidence that sublancin is not only a lantibiotic, but is a Type AIIlantibiotic. The SunT protein supports this conclusion by showing thepresence of the protease domain that is expected for a transporter of aType AII lantibiotic. Moreover, there is strong homology to PepT, whichis a transporter of the lantibiotic Pep5, and lantibiotic transporters(LanT proteins) are generally conserved (2). AII of these considerationsare consistent with sublancin being a lantibiotic of the AII type.

[0026] The evidence that sublancin 168 is a lantibiotic is strong. Thepresublancin gene sequence encodes a serine residue at position 16 ofthe mature region, which can serve as the precursor to dehydroalanine.Sequential Edman degradation was blocked at position 16, which ischaracteristic of dehydro residues. As has been demonstrated for dehydroresidues in other lantibiotics, the block was alleviated byderivatization with ethanethiol. Sequence analysis of the sublancin geneshowed a leader segment with homologies to known Type II lantibiotics,including the “double-glycine” sequence motif immediately preceding thecleavage site, indicating that it is probably translocated by adual-function ABC transporter that both translocates the peptide andproteolytically cleaves the leader segment. The gene immediatelydownstream from the sublancin gene confirms this, in that it encodes aprotein that is homologous to known dual-function transporters, with anidentifiable proteolytic domain in addition to a transporter domain.Although the presublancin gene encodes five cysteines, reaction ofsublancin with an alkylating agent failed to demonstrate the presence ofa free sulfhydryl group, which is consistent with at least one of thecysteines having reacted with a dehydrobutyrine residue to form aP-methyllanthionine bridge. The spectrum of activity of sublancin issimilar to other lantibiotics in that it is active against a variety ofgram-positive bacteria and inactive against gram negative bacteria. Italso showed strong inhibition of bacterial spore outgrowth in additionto Inhibition of exponentially-growing cells, as is seen with both nisinand subtilin. However, unlike nisin and subtilin, washingsublancin-inhibited spores could cause a small percentage (about 1%) ofthem to proceed through outgrowth and then grow vegetatively, suggestingthat the inhibitory effect of sublancin against spores is slightlyreversible. For both nisin and subtilin, it has been demonstrated thatthe mechanism of inhibition of spore outgrowth is different from theinhibition of vegetative growth, in that an intact dehydroalanine isrequired for spore outgrowth inhibition, but not for vegetative growthinhibition. The fact that sublancin contains only one dehydro residuecompared to the three dehydro residues in nisin and subtilin may accountfor sublancin showing reversibility of inhibition of spore outgrowth,whereas nisin and subtilin do not. It has been suggested that thedehydro residue can react with a nucleophilic target (2,27,29), in whichcase the larger number of possible attachment points of nisin andsubtilin could reduce the likelihood of dissociation and reversal ofinhibition, although this explanation is hypothetical.

[0027] With this report of the discovery and characterization ofsublancin 168, the family of known lantibiotics increases in both sizeand scope, and there are now over 20 known lantibiotics (2). A strikingfeature of lantibiotics is their diversity in terms of structure,chemical properties, and biological properties (1,2). The definingcharacteristic of lantibiotics is that they contain the unusual aminoacid lanthionine or β-methyllanthionine, which are formed byposttranslational dehydration of serine or threonine, respectively,followed by a Michael-type nucleophilic addition of a cysteinesulfhydryl across the double bond. Because of this mechanism, thepresence of the lanthionine requires that the cell possess the machineryto dehydrate serines and/or threonines in addition to the ability toform the thioether linkage. Reflecting this, all the currently knownlantibiotics possess at least one lanthionine and one dehydro residue inthe mature peptide, although there is little reason to believe thatexceptions to this are impossible. Especially notable is that, prior tomy discovery of sublancin, all the cysteine residues in knownlantibiotics had undergone posttranslational modifications, and neverexisted as disulfide bridges or free sulfhydryl groups. Sublancin breaksthis trend in that only one of its five cysteines has beenposttranscriptionally modified, and the other four cysteines insteadparticipate in two disulfide bridges.

[0028] Lantibiotics can be considered as a subset of the prodigiousnumber of ribosomally-synthesized antimicrobial peptides that have beendiscovered recently, many of which are produced by eukaryotic organisms,such as the defensins and cecropins (1,32). Mammalian and insectdefensins, tachyplesins, and plant thionins all tend to bedisulfide-rich, typically containing two or three disulfide bridgeswithin a peptide consisting of 30-40 amino acid residues (33). Theubiquity and frequency of disulfide bridges argues an important role,perhaps by their ability to impose conformational constraints on thepeptide and contribute to conformational and chemical stability. Becausethe thioether of the lanthionine bridge contains one sulfur atom insteadof two, the lanthionine would be expected to be moreconformationally-constrained than the disulfide. Moreover, thelanthionine is insensitive to redox conditions, while the disulfide iseasily broken under mild reducing conditions. In view of the apparentsuperiority of the lanthionine bridge in terms of conformational andchemical stability, it is somewhat surprising that sublancin containsone lanthionine and two disulfides, instead of the three lanthioninesand no disulfides that are found in other lantibiotics such as subtilin,which is produced by Bacillus subtilis ATCC 6633, and nisin. The factthat sublancin possesses both types of linkages suggests that havingboth types confers a selective advantage. It has been observed thatantimicrobial peptides represent a remarkable example of convergentevolution, in which a wide variety of organism types have evolvedantimicrobial peptides of common function from very different ancestralorigins (33). Perhaps sublancin represents a converging evolutionarybranch-point between prokaryotic lantibiotics and eukaryotic defensins,in which sublancin has taken advantage of both types of linkages.

[0029] The peptides of the invention may be incorporated into acomposition suitable for killing or inhibiting growth of bacteria,preferably gram-positive bacteria. The peptides are provided incombination with a carrier. Suitable carriers are well known in the art.The processes of producing the compositions of the invention are wellwithin the ordinary skill of a worker in the art, and will therefore notbe described in detail.

[0030] Another useful composition of the invention comprises thepeptides of the invention and another active agent, i.e., anotherlantibiotic or a known antibiotic. Nisin and subtilin are preferredlantibiotics. The combination of ingredients is quite effective whenapplied together to kill or inhibit the growth of bacteria, especiallygram-positive bacteria.

[0031] The peptides of the invention may be incorporated into apharmaceutical preparation suitable for treating a bacterial infectionThe peptides are provided in combination with a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriers arewell known in the art. The processes of producing the pharmaceuticalcompositions of the invention are well within the ordinary skill of aworker in the art, and will therefore not be described in detail.

[0032] The invention includes a method of treating a bacterial infectionin a patient in need thereof, comprising administering to the patient abacterial-infection-treating effective amount of the peptides of theinvention. The peptides may be administered by any acceptable route:oral, intravenous, intraperitoneal, topical, nasal, anal or vaginal. Thedosage range contemplated is 0.01 to 1000 mg/kg body weight in 1-10divided doses. Preferred dosages are 0.1 to 500 mg/kg body weight in 1-6divided doses, more preferred 1.0 to 250 mg/kg body weight in 1-4divided doses.

[0033] It is preferred that the bacteria to be treated is agram-positive bacteria.

[0034] The invention also includes a method of inhibiting bacterialgrowth in a food, comprising adding to the food abacterial-growth-inhibiting effective amount of the peptides of theinvention. A preserved food, comprising a food and abacterial-growth-inhibiting effective amount of the peptides, is alsoincluded in the invention. The amount of the peptides of the inventionto be added are within the range of 0.001 to 1000 mg/kg of food,preferably 0.01 to 800 mg/kg of food, and more preferably 0.1 to 500mg/kg of food.

[0035] It is preferred that the bacteria is a gram-positive bacteria.

[0036] The invention will now be described in reference to the followingnon-limiting examples.

EXAMPLE 1

[0037] Sublancin was isolated from Bacillus subtilis BR151, which is B.subtilis 168 (lys-3 metB10 trpC2), obtained from the Bacillus GeneticsStock Center, Ohio State University, Columbus Ohio. Stocks weremaintained on agar with PAB (17.5 g Bacto antibiotic medium 3 perliter). Sublancin was produced by inoculating 1 L of Medium A with 10 mLof BR151 cultured for 16 hr at 37° C. with vigorous aeration. Medium Ais as previously described (10, 11), except it contained 2% sucroseinstead of 10% sucrose. The culture was agitated vigorously by shaking500 mL volumes at 200 rpm in 2 L baffled flasks at 37° C. for 28 hr,whereupon the culture usually acquired a pinkish-brown color, a fruityodor, and a pH that had dropped to about 6. Good sublancin productionwas consistently obtained when these events were observed. For reasonsthat are not understood, the color, odor, and pH changes did not alwaysoccur, whereupon sublancin production was usually poor. Similarvariability has been reported for subtilin production in B. subtilisATCC 6633 (10).

[0038] To isolate sublancin 168 the culture was acidified to pH 2.5 withconcentrated phosphoric acid, and centrifuged to remove cells. Thesupernatant was made 1 M in NaCl and then applied, by a peristalticpump, to a hydrophobic interaction column constructed with 25 ml ofToyopearl® Butyl-650 resin (TosoHaas, Montgomeryville, Pa.) that hadbeen equilibrated with 1 M NaCl, 50 mM NaAc, pH 4. Unbound proteins wereeluted with several volumes of the loading buffer, and the sublancin waseluted with 50 mM NaAc, pH 4.0; or alternatively, with 30% acetonitrile.After being lyophilized, the residue was dissolved in a minimum amountof water that contained 0.1% TFA, centrifuged to remove particulates,and applied to an analytical reverse-phase C-18 HPLC column(Rainin/Varian, Walnut Creek, Calif.) in a Hewlett-Packard 1050 HPLCmachine with a diode-array detector. Sublancin was eluted using a 2-stepgradient (solvent A was 0.1% TFA in water, solvent B was 0.1% TFA inacetonitrile), the first step going from 0 to 25% solvent B over 30 min,and the second step going from 25-35% solvent B over 30 min, using a 1.2mL/min flow rate throughout.

[0039] Fractions in the second step were assayed for antimicrobialactivity, active fractions were pooled, lyophilized, and then subjectedto a second round of HPLC purification using the same conditions as thefirst round. The elution profile was monitored to detect the presence ofpeptide, dehydro residues, and aromatic residues, respectively. Duringthe second round of HPLC purification, the activity was associated witha single absorbance peak; which was lyophilized and stored at −20° C. Asshown in FIG. 1. the active peak showed absorbances at 214, 254, and 280nm; and when the active peak was treated with ninhydrin, it gave thepurple color that is characteristic of proteins and peptides. Theantimicrobial substance was named sublancin 168, to connote its being anantimicrobial peptide that is produced by B. subtilis 168.

EXAMPLE 2

[0040] Two methods were employed to assay the activity of sublancin 168;a halo assay on plates, and a liquid assay in culture tubes. Bothmethods used Bacillus cereus T spores as the test organism. 250 mg ofspores, prepared as previously described (12), were suspended in 30 mLof distilled water with a glass homogenizer, heat-shocked at 65° C. for2 hr, centrifuged, and resuspended in 50% ethanol. This suspension wassprayed onto the surface of Medium A-containing agar plates using aSigma spray unit (St. Louis Mo.). Prior to spraying, 10-20 μL volumes ofserial dilutions of purified sublancin were spotted onto the plate. Theplates were incubated at 37° C. for 5-12 hr to allow germination andgrowth of the spores, and the diameters of the halos caused by sublancininhibition were measured, and the minimum amount of peptide that wasrequired to give an observable halo was noted. For the liquid assay,heat-shocked spores were suspended in sterile water to a finalconcentration of 2 mg/mL, and then added to culture tubes containing 1%Bacto-tryptone, 0.1 M Tris-Pi, pH 6.8. Prior to adding the spores,serial dilutions of sublancin were added to the tubes. The finalconcentration of spores was 0.1 mg/mL. The tubes were incubated at 37°C. for 3 hr, using sufficient shaking to keep the spores well suspended.The cultures were then examined using phase-contrast microscopy toobserve the extent to which the spores had undergone germination,outgrowth, and vegetative growth. The amount of sublancin required toprevent the spores from proceeding through outgrowth was noted.

EXAMPLE 3

[0041] The lantibiotic family of antimicrobial peptides showsbroad-spectrum activity against gram-positive bacteria, and very littleactivity against gram-negative bacteria (1,2). The ability of sublancinto inhibit growth of a variety of gram-positive and gram-negativebacterial strains was assessed using an agar-diffusion method, and forthose strains that showed sensitivity to sublancin, a minimum inhibitoryconcentration (MIC) was determined. For the agar diffusion test, agarplates contained Difco brain heart infusion (Listera monocytogenes,Lactococcus lactis, Enterococcus faecalis, Steprtococcus pyrogenes) orDifco nutrient broth (Bacillus cereus T, Bacillus megaterium, Bacillussubtilis, Staphylococcus aureus, Staphylococcus epidermidis, Bordetellabronchiseptica, Escherichia coli, Yersinia enterocolitica). A 1,000-folddilution of exponential cultures of the respective strains was made intomolten top agar containing the appropriate medium, which was poured ontothe agar plates. After solidification of the top agar, wells were madewith an Ouchterlony punch and filled with a 20 μL (25 μg) of sublancinsolution. The plates were incubated for 24 hr at 37° C., and thediameters of any halos of inhibition around the wells were measured. Forthose strains that showed a halo of inhibition, a MIC was determined bymaking a 100-fold dilution of an exponential culture of cells in tubescontaining growth medium together with different concentrations (5, 10,25, 50, or 100 μg/mL) of sublancin, which were incubated with shaking at37° C. for 18-30 hr, until the respective control cultures withoutsublancin reached saturation. The MIC was that concentration ofsublancin that completely suppressed growth of the cells.

[0042] Thus, the ability of sublancin to inhibit growth of the batteryof gram-positive and gram-negative bacterial species was tested by themethods used by Cleeland and Squires (26) to evaluate the spectrum ofactivity of antimicrobial agents. As described Infra the strains werefirst assayed for susceptibility to sublancin in an agar-diffusion test.Next, the MIC for susceptible strains was determined in liquid culture.The results in Table I show that the antibiotic spectrum of sublancin isconsistent with its being a lantibiotic, in that inhibition was observedonly among gram-positive strains of bacteria. However, not all thetested strains of gram-positive bacteria were sensitive, and those thatwere sensitive varied considerably in their sensitivity to sublancin.Whereas Bacillus megaterium 14581 and Bacillus subtilis 6633 wereinhibited by 5 μg/mL of sublancin, Bacillus cereus T and Staphylococcusaureus 12600 required more than 100 μg/mL for complete inhibition tooccur. TABLE 1 sensitivity to MIC sublancin (μg/mL) gram positivestrains Bacillus cereus T + >100 Bacillus megaterium (# 14581) ++++ 5Bacillus subtilis (# 6633) ++++ 5 Enterococcus faecalis (# 19433) −Lactococcus lactis (# 11454) − Listeria monocytogenes (# 15313) −Staphylococcus aureus (# 12600) ++ >100 Staphylococcus epidermidis (#12228) − Streptococcus pyogenes (# 49399) ++ 100 gram-negative strainsBordetella bronchiseptica (# 10580) − Escherichia coli JM101 −Pseudomonas aeruginosa (# 10145) − Yersinia enterocolitica (# 23715) −

[0043] Strains were obtained from the American Type Culture Collection(ATCC) in Rockville, Md., and the ATCC strain numbers are indicated.Sensitivity to sublancin was determined by the agar diffusion test, andthe degree of sensitivity was estimated from the diameter of the halo.(++++) indicates a halo diameter>200 mm, (+++) a diameter of 100-200 mm,(+) a diameter<60 mm, and (−) indicates no halo of inhibition. MICvalues indicate the concentrations of sublancin that gave completeinhibition of cell growth in liquid culture.

EXAMPLE 4

[0044] The ability of sublancin to inhibit bacterial spore outgrowth wasalso determined. It had been earlier demonstrated that an intact Dha₅residue in both subtilin (27) and nisin (28) is required in order forinhibition of bacterial spore outgrowth to occur. The fact that anintact Dha₅ residue its unnecessary to inhibit exponentially-growingcells established that the mechanism by which subtilin and nisin inhibitspores is different than the mechanism by which they inhibit growingcells (27). The ability of sublancin to inhibit bacterial sporeoutgrowth was tested using the same methods as for subtilin (27), whichincluded a halo assay in which an agar plate was sprayed with asuspension of B. cereus T spores, and dilutions of sublancin werespotted onto the plate, which was incubated to permit the spores togerminate, outgrow, and grow exponentially to make a confluent lawn.Clear zones occur where the sublancin has been able to inhibit thedevelopment of spores into vegetative cells. The other method was toincubate dilutions of sublancin with spores suspended in growth medium,and use phase-contrast microscopy to observe the stage of inhibition.The latter method established that sublancin permits the spores togerminate, to change from the phase-bright dormant state to thegerminated phase-dark stage; whereupon further development (swelling,elongation, emergence, division) are inhibited (data not shown). In thisliquid assay, the concentration of sublancin required to inhibit sporeoutgrowth was about 0.1 μg/mL, (27 nM) which is significantly less thanthe concentration of nisin (40 nM) or subtilin (80 nM) that is requiredto inhibit outgrowth of these same spores (27,29). It is notable thatsublancin is about 1,000-fold more effective in inhibiting sporeoutgrowth than in inhibiting the same cells in exponential growth. Thecorresponding ratio for subtilin is 30-fold, which means that, althoughsublancin is slightly better at inhibiting spore outgrowth than issubtilin, subtilin is substantially better at killing the correspondingexponentially-growing cells than is sublancin. Using two or morelantibodies, optionally with one or more known antibiotics (e.g.,sublancin and nisin; sublancin and subtilin; or sublancin, nisin andsubtilin) in combination would thus be quite effective. The requirementfor an intact Dha₅ residue in nisin and subtilin in order to exhibitsporostatic activity suggests that the Dha₁₆ residue of sublancin mayalso play an important role in the sporostatic processes.

[0045] While examining the halos caused by sublancin on the lawns ofcells produced by spraying the plate with spores, we noted a discrepancybetween the appearance of the halos produced by sublancin and the halosproduced by either nisin or subtilin (data not shown). With nisin andsubtilin, extended incubation of the plates for several days did notresult in any change in the size or appearance of the halos, whichremained completely clear. In contrast, incubation of the plates thatcontained sublancin halos resulted in occasional colonies growing up inwithin the halos, and a tendency for the surrounding cells to encroachacross the perimeter of the clear zone, to cause the size of the halo todiminish slightly with time. The fact that the sublancin halosdiminished in size whereas the nisin and subtilin halos did not, isexplained by the relatively poor activity of sublancin againstvegetative B. cereus T cells, so once the spores had developed intovegetative cells, they were able to encroach into the halo. However, theappearance of colonies within the clear zone suggested something else,which is that a small fraction of the spores that had been inhibited bysublancin at the post-germination stage were able to overcome thisinhibition and proceed through outgrowth to the vegetative stage. If so,this is in contrast to nisin or subtilin, both of which have been shownto bind and inhibit spores irreversibly (30). To determine whethersublancin binding and inhibition to germinated spores is reversible, theB. cereus T spores were germinated for 3 hr in the presence of variousconcentrations of sublancin ranging from 0.1 to 100 μg/mL, centrifugingthe inhibited spores out of the culture, and resuspending the spores infresh medium without sublancin. The washed spores were then incubatedfor an additional 2-6 hr and examined by phase-contrast microscopy.Whereas most of the spores remained unchanged, a small percentage (about1%) were clearly proceeding through outgrowth, and eventually reachedthe vegetative stage and proliferated. This result is consistent withthe appearance of colonies within the halos on the plates, where thecolonies represent those inhibited spores that recovered after thesublancin had diffused away. The concentration of sublancin used totreat the spores prior to washing had no effect on the outcome of therecovery experiment, with the 0.1 μg/mL treatment showing the sameeffect as the 100 μg/mL treatment. This shows that the spore sites towhich the sublancin become associated are saturated at very low levelsof sublancin.

EXAMPLE 5

[0046] Antimicrobial peptides that are chemically stable are bettersuited for practical applications than are unstable ones. The chemicalstability of sublancin was therefore assessed when it was an unpurifiedcomponent of the culture supernatant, and after it had been purified byHPLC chromatography. Activity was assessed using the agar-plate haloassay against bacterial spores. Culture supernatant stored at roomtemperature showed little change in halo size during the first fourdays, but showed significant loss after 1 week. Culture supernatantsstored at either 4° C. or −20° C. showed no change in halo size after 6months. HPLC-purified sublancin was remarkably stable, and one samplewas stored as a 10 mg/mL solution of sublancin in sterile D₂O, pH 6.5,in an NMR tube for 2 years (protected from light); after which itsactivity remained undiminished and its NMR profile unchanged (data notshown). Sublancin was stable to a wide range of pH values when eitherphosphoric acid or ammonium acetate buffers were used to adjust the pHof culture supernatants over a range of 1.5 to 9.5. The samples wereassayed after incubating them for 2 hr at 4° C. The pH 9.5 halo wasdiminished slightly, but the halos produced by the lower pH samples wereunchanged. Finally, a sample of the culture supernatant that wasautoclaved for 3 min at 121° C. showed undiminished activity. Thesestability characteristics resemble those of nisin, which is very stableat low pH and can survive autoclaving at pH 2.5 without damage, but isfairly unstable above pH 7 (31). However, the ability of sublancin tosurvive in aqueous solution, at a pH that is nearly neutral, for 2 yearswithout any apparent chemical or biological degradation shows that it isa peptide whose intrinsic stability is extremely high. Thisextraordinary stability may prove to be a useful characteristic, perhapsenhancing the utility of sublancin in practical applications, or as amodel compound whose study may inspire strategies for enhancing thestabilities of non-sublancin antimicrobial peptides.

EXAMPLE 6

[0047] For sublancin to be a typical lantibiotic, it should contain atleast one lanthionine residue, either Lan or MeLan; and at least onedehydro residue, either Dha formed from serine, or Dhb formed fromthreonine. The putative mature region of sublancin contains only oneserine (residue 16), and one threonine (residue 19). For sublancin tocontain at least one dehydro residue and one lanthionine residue wouldrequire that both the Ser₁₆ and Thr₁₉ be converted to Dha₁₆ and Dhb₁₉,respectively, and for one of them to form a cross-linkage with acysteine, and for the other to remain as a dehydro residue. Thesepossibilities can be distinguished by NMR spectroscopy. Both Dha and Dhbcontain vinyl protons, which typically give resonance peaks in theδ=5.2-6.9 ppm region of the NMR spectrum, with Dha appearing as adoublet, and Dhb appearing as a quartet (7,21-23). The NMR spectrum ofsublancin is shown in FIG. 6. A portion of the NMR spectrum shows adoublet appearing at δ6.2 ppm which is in the middle of the vinyl protonregion, and therefore argues that a dehydro residue is present, and itsbeing a doublet further argues that it is a Dha. The other peaks are inthe aromatic proton region (δ=6.5 to 8.0), and can be attributed to thearomatic residues in sublancin. It is to be noted that the Edmandegradation of native sublancin was blocked from residue 16 on, and thisblock was alleviated by reacting with ethanethiol, which is alsoconsistent with residue 16 being a dehydro residue. Since the genesequence shows a Ser at position 16, one can conclude that the Dha shownin the NMR spectrum is derived by post-translational dehydration ofSer₁₆ to Dha₁₆.

EXAMPLE 7

[0048] When sublancin was subjected to SDS-PAGE, it showed a single bandthat migrated at a position that corresponded to a molecular mass ofapproximately 4 kDa (data not shown). Ion-spray mass spectroscopyprovided a more precise molecular mass of 3877.78 kDa as shown inFIG. 1. The sublancin molecular mass as predicted from the amino acidsequence encoded in the sublancin gene is 3713.3 Da, assuming one MeLan,one Dha, and four cysteines existing in two disulfide bridges. There isthus a discrepancy of 164.48 Da between this predicted molecular massand the actual molecular mass, which may be due to one or moreadditional modifications of the amino acids.

[0049] Purified sublancin was sequenced from its N-terminal end usingEdman degradation, using an Applied Biosystems (Foster City, Calif.)Model 477A peptide sequencer with an on-line HPLC analyzer in theUnivesity of Maryland Core Facility (Baltimore, Md.). Amino acidcomposition analysis was performed on HCl hydrolysates by CommonwealthBiotechnologies, Inc. (Richmond Va.). Sublancin was treated withethanethiol in order to sequence through any dehydro residues, which areotherwise blocked, using the method of Meyer, et al. (13). Themodification mixture consisted of 280 μL ethanol, 200 μL steriledeionized water, 65 μL of 5 M NaOH, and 60 μL ethanethiol. 150 μL ofthis modification mixture was added to 50 μg of freeze-dried sublancinand incubated under nitrogen for 1 hr at 50° C. The pH was lowered byaddition of 5 μL of glacial acetic acid, and the product purified byHPLC as described above for sublancin.

[0050] Sequence analysis using Edman degradation yielded a sequence ofGly-Leu-Gly-Lys-Ala-Gln-blank-Ala-Ala-Leu-Trp-Leu-Gln-blank-Ala-blank-blank-blank.The blank cycles were those that did not show an identifiable aminoacid, some of which could be due to Cys residues, which wereunderivatized and therefore not detectable. Other potential sources ofblank cycles are the unusual amino acid residues typically found inlantibiotics. For example, lanthionine residues do not produce peaksthat are identifiable as normal amino acids, and the dehydro residuesblock the sequence analysis because they spontaneously lose theirN-terminal amino group and are therefore unable to react with the Edmanreagent (17), thus bringing the sequence analysis to a halt. Thisdehydro-residue block can be alleviated by reacting the peptide withethanethiol, which adds across the double bond, thus preventing loss ofthe N-terminal amino group (13). Sublancin was accordingly derivatizedwith ethanethiol, whereupon it was possible to sequence past theapparent block at position 16, and to obtain Gly both at positions 17and 18; but then a blank was encountered at position 19. The fact thatethanethiol derivatization alleviated the block at position 16 is strongevidence that residue 16 is a dehydro residue.

[0051] From DNA sequencing, it was determined that the sublancinprepeptide contains 5 cysteine residues, which is the same number ofcysteines as are present in the prepeptides of nisin and subtilin.However, in nisin, subtilin, and all other known antibiotics, all of thecysteine residues are converted to unusual residues such as the 5 Lanand MeLan in nisin (24)and subtilin (11), or the aminovinylcysteine inepidermin (25). For a natural lantibiotic to contain unmodifiedcysteines or disulfide cross-linkages is unprecedented, so the cysteineresidues in sublancin were examined to see if any possessed thecharacteristics of either free sulfhydryl groups or disulfide bridges.The amino acid analysis that was employed cannot detect free cysteineresidues, but can detect them as carboxymethyl-cysteine if they arealkylated prior to acid hydrolysis. Alkylation of native sublancinfollowed by amino acid analysis gave no detectablecarboxymethyl-cysteine, which rules out the presence of free sulfhydrylgroups (data not shown). Reduction of sublancin with DTT followed byalkylation gave 3.3 (suggesting a real value of 4, since the 3.3 islikely a minimum value, and the nearest integer value larger than 3.3 is4) carboxymethyl-cysteines per mole of sublancin. SDS-PAGE and ion-spraymass spectroscopy results described above established that sublancinexists exclusively as a monomer, so there cannot he any intermoleculardisulfide bridges. These observations are all consistent with 4 of thecysteines of sublancin participating in two disulfide bridges, with thefifth cysteine having been converted to a MeLan residue by reacting witha Dhb residue (derived from post-translational dehydration of Thr₁₉),leaving the unreacted Dha₁₆ that is revealed in NMR spectrum.

[0052] One dimensional NMR spectroscopy was performed with a BrukerAMX-500 NMR spectrometer interfaced to an Aspect 3000 computer usingUXNMR software. Lyophilized sublancin was dissolved in 99.96% atom % D₂Oto exchange protons and lyophilized (done twice) and dissolved in D₂O toa final concentration of 10 mg/mL. The proton spectra were recorded atconstant 295° K. in D₂O with and without the suppression of the watersolvent resonance. Mass spectral analysis was performed by PeptidoGenicResearch & Co (Livermore, Calif.) on a Sciex API I Electrospray massspectrometer. The reported masses are those calculated as the mostprobable values based on the different m/z forms.

[0053] The NMR spectrum of sublancin showed a doublet in the vinylproton region of the spectrum, which is consistent with the presence ofa dehydroalanine.

EXAMPLE 8

[0054] The number and location of disulfide bridges was further exploredby analysis with. proteolytic enzymes. The native form of sublancin, andthe denatured form, and the denatured-reduced form of sublancin were allresistant to trypsin, despite the presence of a Lys at position 4 and anArg at position 33. When the denatured and reduced sublancin wasalkylated, trypsin cleavage gave detectable amounts of fragments of3,200 and 1,581 Da, neither of which is an expected product. Sublancinwas more sensitive to chymotrypsin, with even the native molecule beingsubstantially degraded, to give products of 1,392 and 1,823 Da. Thefirst is consistent with a polypeptide consisting of residues 1-11 beingcross linked by a disulfide bridge to a peptide consisting of residues36-37 (G₁-W₁₁-S-S-C₃₆-R₃₇ , with an expected value of 1,392 Da) and thesecond is consistent with a polypeptide consisting of residues 1-11cross linked by a disulfide bridge to a peptide consisting of residues33-37 (G₁-W₁₁-S-S-R₃₃-R₃₇, with an expected value of 1,823 Da); withchymotrypsin having cleaved at typical major cleavage sites (W₁₁, Y₃₂,F₃₅,). From this, we can conclude that native sublancin has a disulfidebridge between Cys₇ and Cys₃₆. To decide upon the location of the seconddisulfide bridge, we compare sublancin to other Type A lantibiotics, andnote that formation of a thioether link between Cys₂₂ and Dhb₁₉, to givea Aba₁₉-Ala₂₂ MeLan-type cross-linkage would put a 2-residue Gly₂₀-Gly₂₁sequence in the ring enclosed by the MeLan cross-link, which is similarto the 2-residue Prog-Gly₁₀ sequence enclosed by the Aba₈-Ala₁₁ MeLancross-link in both nisin and subtilin. Moreover, formation of thisparticular MeLan bond is consistent with the observation that theCys-Dha partner selection in lantibiotics consistently involves adehydro residue that is on the N-terminal side of the Cys residue.Assuming that the MeLan that actually forms conforms to these standardpatterns, then Cys₂₂ will react with Dhb₁₉, which would require thesecond disulfide bridge to form between Cys₁₄ and Cys₂₉, as shown inFIG. 7.

EXAMPLE 9

[0055] Since lantibiotics are biosynthesized from gene-encodedprecursors, one approach to determine if sublancin is a lantibiotic isto see if it is gene-encoded, and if it is, to examine the gene and theoperon in which it is found to see if they possess features that arecharacteristic of lantibiotics. To determine whether sublancin is agene-encoded peptide, the N-terminal sequence was used to design ahybridization probe, which was then used to screen a B. subtilis 168genomic library that had been constructed in bacteriophage λ. Clonescontaining positive signals were subjected to DNA sequence analysis.Nearly 5 kb of sequence was obtained, which we published in a publicdatabase as soon as it was complete (18). This sequence was searched foropen reading frames (ORFs), which were in turn searched for theN-terminal amino acid sequence of sublancin 168. A 56-residue ORF, shownin FIG. 2, that contained a perfect match to the N-terminal sequence ofthe sublancin peptide was found near the center of the 5 kb sequence. Inaddition, a 332-residue ORF was found upstream from the sublancin gene,and about 560 residues of a partially-complete ORF was found downstreamfrom the sublancin gene. The locations of these three ORFs within the 5kb sequence are shown in FIG. 2. Several months after our sequence waspublished in GenBank, the Bacillus Genome Project published the completeB. subtilis 168 genome (9), which mapped these genes at a position of193.80° on the B. subtilis 168 chromosome.

[0056] The putative functions of the upstream and downstream ORFs wereexplored by searching the GenBank/EMBL nucleotide databases forhomologies to proteins with known functions. The 332-residue upstreamORF (denoted uvrX) showed extensive homologies to proteins involved inrepair of u.v. damage to DNA, so a role in the biosynthetic pathway ofsublancin seems unlikely. The 560-residue segment of the downstream ORFshowed homologies to known ABC transporter proteins including PepT,which is the transporter that is responsible for secretion of Pep5during its biosynthesis (19). The gene for this downstream ORF (denotedsunT) is therefore a strong candidate as the corresponding transporterthat participates in the secretion of sublancin. FIG. 3 shows thesegment of the DNA sequence that contains the sublancin gene (sunA), andthe 5-prime end of sunT, together with their conceptual translations(sunA and the N-terminal portion of sunT), the putative promoter regionof the sun operon, and the ribosome binding site of the sunA mRNA. Thecomplete sequences and their conceptual translations are available asAccession Number AF069294 in GenBank.

EXAMPLE 10

[0057] A B. subtilis 168 genomic library was constructed inbacteriophage λ using total chromosomal DNA from strain BR151 grown in50 mL of PAB. Cells were lysed with a mixture of lysozyme, sodiumdodecyl sulfate, and proteinase K; and the DNA was recovered anddeproteinized with phenol-chloroform as previously described (11). Thegenomic DNA was partially-digested with Sau 3Al to give random fragmentsin a 12-23 kb size range, which were cloned into LambdaGEM-12 partiallyfilled-in Xho I half-site arms obtained from Promega (Madison, Wis.),which were then packaged into E. coli cells using the protocol providedby the manufacturer. The library was screened for the sublancin geneusing synthetic DNA oligomers whose sequences were chosen using thestrategy of Lathe (14), based on the 16-residue N-terminal sequence ofsublancin. Three single-sequence 48-mer probes were designed, each onewith randomly-chosen degenerate bases, and the synthesis was performedby Ransom Hill Bioscience (Ramona Calif.). For those amino acid residuesthat appeared as unidentifiable blanks in the sequence, inosines wereplaced in the corresponding codons in the probes. The three probes were:P     r     o       b     e                 1    :GGGTTGGGTAAAGCCCAAIIIGCGGCCTTGTGGTTACAGIIIGCTTCCP     r     o       b     e                 2    :GGGTTGGGCAAAGCACAGIIIGCGGCTTTTTGGTTACAGIIIGCGTGCP     r     o       b     e                 3    :GGACTTGGTAAAGCGCAAIIIGCAGCTCTGTGGCTTCAAIIIGCATGC.

[0058] The probes were radio-labeled with ³²P at their 5-prime endsusing T4 polynucleotide kinase and hybridized to Southern blots ofrestriction digests of BR151 genomic DNA under a variety of temperatureand ionic strength conditions in order to optimize the signal strengthand specificity. Probe 1 gave a good signal when hybridized at 45° C. in6× SSC and washed at 37° C. in 2× SSC; whereas probe 3 gave a goodsignal when hybridized at 45° C. in 6× SSC and washed at 45° C. in 2×SSC. A good signal for probe 2 could not be obtained, so its use wasabandoned. The bacteriophage λ library was plated and transferred toduplicate nitrocellulose filters using standard procedures (15). One ofthe duplicate filters was hybridized to probe 1, and the other to probe3. The only plaques selected for further study were those thathybridized to both probes. Several such dual-hybridizing plaques werepicked, their inserts were subcloned into pTZ plasmids and screenedagain with probes 1 and 3. Positive inserts were cloned into M13 andsubjected to dideoxy sequence analysis. The DNA sequences wereconceptually translated into six reading frames, which were searched forthe N-terminal amino acid sequence of sublancin. When the sublancinsequence was found, the actual DNA sequence that encoded the sublancingene could be identified, which provided the sequence information neededto synthesize probes that were exactly homologous to the sublancin gene.These were used to identify library clones that contained sequences thatsurrounded the subtilin gene, which were then also subcloned andsequenced.

[0059] References

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1 18 1 48 DNA Artificial Sequence modified_base (19) adenine is modifedto inosine 1 gggttgggta aagcccaann ngcggccttg tggttacagn nngcttcc 48 248 DNA Artificial Sequence modified_base (19) adenine is modifed toinosine 2 gggttgggca aagcacagnn ngcggctttt tggttacagn nngcgtgc 48 3 48DNA Artificial Sequence modified_base (19) adenine is modifed to inosine3 ggacttggta aagcgcaann ngcagctctg tggcttcaan nngcatgc 48 4 792 DNABacillus subtilis 168 CDS (219)..(389) CDS (447)..(782) 4 attattaattcaaaataaat ccataatagt caattttatt tagtgtatta caaccaattc 60 tgtttattgataggtaataa agtttttttt ctatgattta tgaacaagtt tccttataat 120 tttcaaaaaaaaataaaaaa tatggttgaa tttagattta tcttccttta tattaaaaaa 180 tgtaatccggattgcaaaca aatggggagg ttttacaa atg gaa aag cta ttt aaa 236 Met Glu LysLeu Phe Lys 1 5 gaa gtt aaa cta gag gaa ctc gaa aac caa aaa ggt agt ggatta gga 284 Glu Val Lys Leu Glu Glu Leu Glu Asn Gln Lys Gly Ser Gly LeuGly 10 15 20 aaa gct cag tgt gct gcg ttg tgg cta caa tgt gct agt ggc ggtaca 332 Lys Ala Gln Cys Ala Ala Leu Trp Leu Gln Cys Ala Ser Gly Gly Thr25 30 35 att ggt tgt ggt ggc gga gct gtt gct tgt caa aac tat cgt caa ttc380 Ile Gly Cys Gly Gly Gly Ala Val Ala Cys Gln Asn Tyr Arg Gln Phe 4045 50 tgc aga taa aacatttgta gagggaatat tttaaatatt ccctcatatt 429 CysArg 55 taaagcgggg attgaaa ttg aat aag aaa aag aaa tat gtt cat act aaa479 Met Asn Lys Lys Lys Lys Tyr Val His Thr Lys 60 65 cag ttt aat agtcat gat tgt gga cta gct tgt atc tcg tca att tta 527 Gln Phe Asn Ser HisAsp Cys Gly Leu Ala Cys Ile Ser Ser Ile Leu 70 75 80 aag ttt cat aac cttaac tat gga att gat ttc tta cta gac cta att 575 Lys Phe His Asn Leu AsnTyr Gly Ile Asp Phe Leu Leu Asp Leu Ile 85 90 95 100 ggg gat aag gaa ggctat agt tta aga gac tta att gtt att ttt aag 623 Gly Asp Lys Glu Gly TyrSer Leu Arg Asp Leu Ile Val Ile Phe Lys 105 110 115 aag atg ggg ata aaaact agg cca ctt gaa ttg caa gaa aat aag aca 671 Lys Met Gly Ile Lys ThrArg Pro Leu Glu Leu Gln Glu Asn Lys Thr 120 125 130 ttc gaa gcc cta aaacaa ata aag ctc cct tgt ata gct ttg tta gaa 719 Phe Glu Ala Leu Lys GlnIle Lys Leu Pro Cys Ile Ala Leu Leu Glu 135 140 145 ggg gag gaa tat ggacat tac ata aca ata tac gaa att aga aat aac 767 Gly Glu Glu Tyr Gly HisTyr Ile Thr Ile Tyr Glu Ile Arg Asn Asn 150 155 160 tat tta ctt gtt agtgatcctgata 792 Tyr Leu Leu Val Ser 165 5 56 PRT Bacillus subtilis 168Pre-Sublancin 168 5 Met Glu Lys Leu Phe Lys Glu Val Lys Leu Glu Glu LeuGlu Asn Gln 1 5 10 15 Lys Gly Ser Gly Leu Gly Lys Ala Gln Cys Ala AlaLeu Trp Leu Gln 20 25 30 Cys Ala Ser Gly Gly Thr Ile Gly Cys Gly Gly GlyAla Val Ala Cys 35 40 45 Gln Asn Tyr Arg Gln Phe Cys Arg 50 55 6 112 PRTBacillus subtilis 168 6 Met Asn Lys Lys Lys Lys Tyr Val His Thr Lys GlnPhe Asn Ser His 1 5 10 15 Asp Cys Gly Leu Ala Cys Ile Ser Ser Ile LeuLys Phe His Asn Leu 20 25 30 Asn Tyr Gly Ile Asp Phe Leu Leu Asp Leu IleGly Asp Lys Glu Gly 35 40 45 Tyr Ser Leu Arg Asp Leu Ile Val Ile Phe LysLys Met Gly Ile Lys 50 55 60 Thr Arg Pro Leu Glu Leu Gln Glu Asn Lys ThrPhe Glu Ala Leu Lys 65 70 75 80 Gln Ile Lys Leu Pro Cys Ile Ala Leu LeuGlu Gly Glu Glu Tyr Gly 85 90 95 His Tyr Ile Thr Ile Tyr Glu Ile Arg AsnAsn Tyr Leu Leu Val Ser 100 105 110 7 37 PRT Bacillus subtilis 168Prosublancin 168 7 Gly Leu Gly Lys Ala Gln Cys Ala Ala Leu Trp Leu GlnCys Ala Ser 1 5 10 15 Gly Gly Thr Ile Gly Cys Gly Gly Gly Ala Val AlaCys Gln Asn Tyr 20 25 30 Arg Gln Phe Cys Arg 35 8 57 PRT Unknown Nisin A8 Met Ser Thr Lys Asp Phe Asn Leu Asp Leu Val Ser Val Ser Lys Lys 1 5 1015 Asp Ser Gly Ala Ser Pro Arg Ile Thr Ser Ile Ser Leu Cys Thr Pro 20 2530 Gly Cys Lys Thr Gly Ala Leu Met Gly Cys Asn Met Lys Thr Ala Thr 35 4045 Cys His Cys Ser Ile His Val Ser Lys 50 55 9 56 PRT Unknown subtilin 9Met Ser Lys Phe Asp Asp Phe Asp Leu Asp Val Val Lys Val Ser Lys 1 5 1015 Gln Asp Ser Lys Ile Thr Pro Gln Trp Lys Ser Glu Ser Leu Cys Thr 20 2530 Pro Gly Cys Val Thr Gly Ala Leu Gln Thr Cys Phe Leu Gln Thr Leu 35 4045 Thr Cys Asn Cys Lys Ile Ser Lys 50 55 10 52 PRT Unknown epidermin 10Met Glu Ala Val Lys Glu Lys Asn Asp Leu Phe Asn Leu Asp Val Lys 1 5 1015 Val Asn Ala Lys Glu Ser Asn Asp Ser Gly Ala Glu Pro Arg Ile Ala 20 2530 Ser Lys Phe Ile Cys Thr Pro Gly Cys Ala Lys Thr Gly Ser Phe Asn 35 4045 Ser Tyr Cys Cys 50 11 60 PRT Unknown Pep5 11 Met Lys Asn Asn Lys AsnLeu Phe Asp Leu Glu Ile Lys Lys Glu Thr 1 5 10 15 Ser Gln Asn Thr AspGlu Leu Glu Pro Gln Thr Ala Gly Pro Ala Ile 20 25 30 Arg Ala Ser Val LysGln Cys Gln Lys Thr Leu Lys Ala Thr Arg Leu 35 40 45 Phe Thr Val Ser CysLys Gly Lys Asn Gly Cys Lys 50 55 60 12 51 PRT Unknown Lacticin 481 12Met Lys Glu Gln Asn Ser Phe Asn Leu Leu Gln Glu Val Thr Glu Ser 1 5 1015 Glu Leu Asp Leu Ile Leu Gly Ala Lys Gly Gly Ser Gly Val Ile His 20 2530 Thr Ile Ser His Glu Cys Asn Met Asn Ser Trp Gln Phe Val Phe Thr 35 4045 Cys Cys Ser 50 13 51 PRT Streptococcus pyogenes Streptococcin A-FF2213 Met Glu Lys Asn Asn Glu Val Ile Asn Ser Ile Gln Glu Val Ser Leu 1 510 15 Glu Glu Leu Asp Gln Ile Ile Gly Ala Gly Lys Asn Gly Val Phe Lys 2025 30 Thr Ile Ser His Glu Cys His Leu Asn Thr Trp Ala Phe Leu Ala Thr 3540 45 Cys Cys Ser 50 14 51 PRT Unknown Salivaricin A 14 Met Asn Ala MetLys Asn Ser Lys Asp Ile Leu Asn Asn Ala Ile Glu 1 5 10 15 Glu Val SerGlu Lys Glu Leu Met Glu Val Ala Gly Gly Lys Arg Gly 20 25 30 Ser Gly TrpIle Ala Thr Ile Thr Asp Asp Cys Pro Asn Ser Val Phe 35 40 45 Val Cys Cys50 15 68 PRT Unknown Cytolysin L1 15 Met Glu Asn Leu Ser Val Val Pro SerPhe Glu Glu Leu Ser Val Glu 1 5 10 15 Glu Met Glu Ala Ile Gln Gly SerGly Asp Val Gln Ala Glu Thr Thr 20 25 30 Pro Val Cys Ala Val Ala Ala ThrAla Ala Ala Ser Ser Ala Ala Cys 35 40 45 Gly Trp Val Gly Gly Gly Ile PheThr Gly Val Thr Val Val Val Ser 50 55 60 Leu Lys His Cys 65 16 181 PRTUnknown LcnDR3 16 Met Lys Ile Val Leu Gln Asn Asn Glu Gln Asn Cys LeuLeu Ala Cys 1 5 10 15 Tyr Ser Met Ile Leu Gly Tyr Phe Gly Arg Asp ValAla Ile His Glu 20 25 30 Leu Tyr Ser Gly Glu Met Ile Pro Pro Asp Gly LeuSer Val Ser Tyr 35 40 45 Leu Lys Asn Ile Asn Met Lys His Gln Val Ser MetHis Val Tyr Lys 50 55 60 Thr Asp Lys Lys Asn Ser Pro Asn Lys Ile Phe TyrPro Lys Met Leu 65 70 75 80 Pro Val Ile Ile Gln Trp Asn Asp Asn His PheVal Val Val Thr Lys 85 90 95 Ile Tyr Arg Lys Asn Val Thr Leu Ile Asp ProAla Ile Gly Lys Val 100 105 110 Lys Tyr Asn Tyr Asn Asp Phe Met Lys LysPhe Ser Gly Tyr Ile Ile 115 120 125 Thr Leu Ser Pro Asn Ser Ser Phe ThrLys Lys Lys Arg Ile Ser Glu 130 135 140 Ile Ile Phe Pro Leu Lys Lys IlePhe Lys Asn Arg Asn Thr Phe Leu 145 150 155 160 Tyr Ile Phe Ser Leu PheIle Ser Gln Ile Val Ala Leu Trp Phe Ser 165 170 175 Ile Ile Leu Arg Asp180 17 47 PRT Unknown PepT 17 Met Lys Lys Glu Asn Pro Leu Phe Phe LeuPhe Ser Lys Ile Lys Trp 1 5 10 15 Pro Lys Ser Leu Phe Ile Ile Ala IleIle Ile Ser Ser Ile Gly Ser 20 25 30 Ile Thr Glu Ile Ile Val Pro Leu LeuThr Gly Asn Leu Ile Asp 35 40 45 18 37 PRT Bacillus subtilis 168DISULFID (14)..(29) DISULFID (7)..(36) MOD_RES (16) Xaa is adehydrogenated Ser (Dha) 18 Gly Leu Gly Lys Ala Gln Cys Ala Ala Leu TrpLeu Gln Cys Ala Xaa 1 5 10 15 Gly Gly Xaa Ile Gly Cys Gly Gly Gly AlaVal Ala Cys Gln Asn Tyr 20 25 30 Arg Gln Phe Cys Arg 35

What is claimed is:
 1. A peptide having an amino acid sequence which isat least 80% identical with SEQ ID NO: 7 prior to dehydration of serinesand threonines and formation of thioether cross-linkages.
 2. The peptideof claim 1, wherein the peptide contains the amino acids of positions 7,14, 16, 19, 22, 29 and 36 of SEQ ID NO:
 7. 3. The peptide of claim 2,wherein a thioether cross-linkage is formed between the amino acids ofpositions 19 and 22 and disulfide cross-linkages are formed between theamino acids of positions 7 and 36 and positions 14 and
 29. 4. Thepeptide of claim 1, wherein the amino acid sequence is at least 90%identical with SEQ ID NO:
 7. 5. The peptide of claim 1, wherein theamino acid sequence is 100% identical with SEQ ID NO:
 7. 6. A peptidehaving an amino acid sequence which is at least 80% identical with SEQID NO: 5 prior to dehydration of serines and threonines and formation ofthioether cross-linkages.
 7. The peptide of claim 6, wherein the aminoacid sequence is at least 90% identical with SEQ ID NO:
 5. 8. Thepeptide of claim 6, wherein the amino acid sequence is 100% identicalwith SEQ ID NO:
 5. 9. A peptide having an amino acid sequence which isat least 80% identical with SEQ ID NO:
 18. 10. The peptide of claim 9,wherein the peptide contains the amino acids of positions 7, 14, 16, 19,22, 29 and 36 of SEQ ID NO:
 18. 11. The peptide of claim 10, wherein athioether cross-linkage is formed between the amino acids of positions19 and 22 and disulfide cross-linkages are formed between the aminoacids of positions 7 and 36 and positions 14 and
 29. 12. The peptide ofclaim 9, wherein the amino acid sequence is at least 90% identical withSEQ ID NO:
 18. 13. The peptide of claim 9, wherein the amino acidsequence is 100% identical with SEQ ID NO:
 18. 14. A pharmaceuticalpreparation suitable for treating a bacterial infection, comprising thepeptide of claim 9 in combination with a pharmaceutically acceptablecarrier.
 15. A preserved food, comprising a food and abacterial-growth-inhibiting effective amount of the peptide of claim 9.16. A method of treating a bacterial infection in a patient in needthereof, comprising administering to the patient abacterial-infection-treating effective amount of the peptide of claim 9.17. The method of claim 16, wherein the bacteria is a gram-positivebacteria.
 18. A method of inhibiting bacterial growth in a food,comprising adding to the food a bacterial-growth-inhibiting effectiveamount of the peptide of claim
 9. 19. The method of claim 18, whereinthe bacteria is a gram-positive bacteria.
 20. A DNA having a nucleicacid sequence which is at least 80% identical with nucleotides 219-389of SEQ ID NO:
 4. 21. The DNA of claim 20, wherein the nucleic acidsequence is at least 90% identical with nucleotides 219-389 of SEQ IDNO:
 4. 22. The DNA of claim 20, wherein the nucleic acid sequence is100% identical with nucleotides 219-389 of SEQ ID NO:
 4. 23. A DNAhaving a nucleic acid sequence which is at least 80% identical withnucleotides 447-782 of SEQ ID NO:
 4. 24. The DNA of claim 20, whereinthe nucleic acid sequence is at least 90% identical with nucleotides447-782 of SEQ ID NO:
 4. 25. The DNA of claim 20, wherein the nucleicacid sequence is 100% identical with nucleotides 447-782 of SEQ ID NO:4.
 26. A composition suitable for killing or inhibiting growth ofbacteria, comprising the peptide of claim 9 and a carrier.
 27. Acomposition suitable for killing or inhibiting growth of bacteria,comprising the peptide of claim 9 and a second active agent selectedfrom the group consisting of a lantibiotic and an antibiotic.
 28. Thecomposition of claim 27, wherein the second active agent is nisin. 29.The composition of claim 27, wherein the second active agent issubtilin.